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CRISPR- a New Tool for DNA Editing

CRISPR – a powerful DNA editing technology has been named Science magazine’s 2015 “Breakthrough of the Year.”

If you haven’t already heard of it, it’s a good time to do some web surfing, because CRISPR, short for clustered regularly-interspaced short palindromic repeats, is predicted to change the face of medicine and, some say, “the world as we know it.”

According to Dr David King, founder and Director of Human Genetics Alert, “This is the first step on a path that scientists have carefully mapped out towards the legalization of genetically-modified babies.”

Dr Fiachra O’Brolcháin, a Marie Curie/Assistid Research Fellow at the Institute of Ethics, Dublin City University, “We might find ourselves inaugurating an era of ‘liberal eugenics’, in which future generations are created according to consumer choice.”

Seth Shostak, Director of the Search for Extraterrestrial Intelligence (SETI) Institute, believes that we will finally understand biology at a molecular level. As a result humanity will be able to cure all diseases and usher in an era of ‘designer humans’.

CRISPR and CRISPR-associated (Cas) genes are essential to adaptive immunity in select bacteria and archaea enabling them to eliminate the genetic material of an invading virus then store bits of the virus’s DNA to be used to defend against future attacks. Cas9 (CRISPR-associated protein 9; Csn1) a part of this defense mechanism found in Streptococcus pyogenes (the bacterium that causes strep throat) has been shown to be a key player in certain CRISPR systems. A genome editing tool based on CRISPR- Cas9 has generated considerable excitement.

CRISPR tells Cas9 exactly where to snip. To achieve site-specific DNA recognition and cleavage Cas9 must be complexed with both a small CRISPR RNA (crRNA) and a separate trans-activating crRNA (tracrRNA) that is partially complementary to the crRNA. During the destruction of the target DNA, both DNA strands are cut, which generates double-stranded breaks (DSB). This occurs only in the presence of a protospacer-associated motif (PAM) sequence above the break and it triggers the activation of the DSB repair machinery, which begins the repair process to restore the strands to their original DNA structure. Conveniently, the genes that encode for Cas are always sitting somewhere near the CRISPR sequences. A number of variants of this system have been developed to simplify and make the process more site-specific.

CRISPR-Cas is a valuable strategy for targeted genomic engineering. Not only can faulty genes be repaired by cutting out bad genetic material and injecting a normal copy into the cell, the tool is seen as a way to induce gene inactivation or modification (useful for producing genetically modified animals for research or drug screening) or the insertion of heterologous genes that can regulate endogenous gene expression or label specific genomic loci in living cells. CRISPR is also being used to perform genome-wide screening, to insert fluorescent genomic loci into cells to visualize genomic elements, and for proteomic analysis of a single genomic locus.

Using a gRNA tailored to a specific gene the CRISPR-Cas system allows the cutting and pasting of bits of DNA sequence into the genome at any point. Not only is the tool relatively simple – it’s cheap and easy to get. Target sequences can be designed and ordered online together with matched gRNA. It takes no longer than a few days for the guide sequence to arrive by mail. The ubiquitous access to and simplicity of creating CRISPRs creates opportunities for scientists, as well as non-scientists, in any part of the world to do any kind of genetic experiments they want. Low cost and ease of obtainability of CRISPR kits have led to “biohackers” (i.e., amateurs) toying with gene rewriting in their garages and kitchens. Researchers often need to order only the RNA fragment; the other components can be bought off the shelf. Total cost: as little as $30.

Besides simplicity and adaptability, CRISPR-Cas systems are reported to have high targeting efficiency, ie, the percentage of desired mutation achieved. Unlike other genomic editing tools, the CRISPR-Cas9 system requires only the redesign of the crRNA to change target specificity. These positive characteristics have made CRISPR-Cas one of the most popular approaches for genome engineering. However, off-target mutations remain a stumbling block for wider use, especially in the clinic. Several groups have developed web based tools (CRISPR Design Tool and ZiFiT Targeter) to facilitate the identification of potential CRISPR target sites and assess their potential for off-target cleavage.

In 2015, Chinese scientists used CRISPR-Cas9 to edit the genome of human embryos for the first time provoking debates about the ethical implications of such work. The purpose of the experiment was to explore the possibility of modifying the gene responsible for β-thalassaemia, a potentially fatal blood disorder, using ‘non-viable’ embryos. Low success rate and a high number of off-target mutations quickly ended the experiment. The resulting paper was rejected by Nature and Science because of ethical concerns. Though the study was seen as a landmark, George Daley, a stem-cell biologist at Harvard Medical School in Boston, Massachusetts, USA said, “Their study should be a stern warning to any practitioner who thinks the technology is ready for testing to eradicate disease genes.”

The debate continues with some scientists affirming that gene editing in embryos could eradicate devastating genetic diseases before a baby is born. Others have expressed concerns that any gene-editing research on human embryos could be a slippery slope towards unsafe or unethical uses of the technique. While most people think it is acceptable to fix faulty genes in somatic cells, the use of this technology in germline tissues (eggs, sperm, or tissues that produce those reproductive cells) that can be passed on to future generations crosses an ethical line for many.

CRISPR has been getting a lot of coverage as a future medical treatment. Last year, bioengineer Daniel Anderson of the Massachusetts Institute of Technology in Cambridge, USA and his colleagues used CRISPR in mice to fix a disease-causing mutation associated with a human metabolic disease called tyrosinaemia. Scientists at Temple University, in Philadelphia, Pennsylvania, USA used CRISPR to snip out integrated HIV-1 genes from human cells.

CRISPR may also have a major impact in ecology and conservation. Researchers at the University of California at Irvine and at San Diego, California, USA have used the CRISPR system to genetically modify mosquitoes to fight malaria in their bodies and pass that trait to 97% of their offspring. Genetic modifications usually take a long time to spread through a population because a mutation carried by one pair of chromosomes is inherited by only half the offspring. But a gene drive (“selfish” gene) allows a mutation made by CRISPR on one chromosome to copy itself to its partner in every generation, so that nearly all offspring will inherit the change. Gene drives use the cell’s own repair machinery to ensure that they show up on both chromosomes in a pair, giving them better than 50-50 odds of being passed on to future generations. This means that it will speed through a population exponentially faster than normal; thus a mutation engineered into a mosquito could spread through a large population within a season. If that mutation also reduced the number of offspring a mosquito produced, then the population could be wiped out, along with any malaria parasites it was carrying.

With CRISPR new genes can be slipped into the drive system to eliminate diseases, create hardier plants, or wipe out pathogens. Potential beneficial uses of such gene drives besides ridding the world of pest-borne diseases like malaria, dengue fever, and Lyme disease include reversing the development of pesticide and herbicide resistance, and locally eradicating invasive species, like rats. However, drives may present environmental and security challenges as well as benefits. Gene drives that have the ability to alter a substantial fraction of a wild population could have unpredictable ecological consequences, such as spreading to other species.

The simplicity, efficiency, and versatility of the CRISPR-Cas system has allowed for rapid progress in developing Cas9 tools for cell and molecular biology research and its use will likely be limited only by our imagination—ethical concerns remain, however. The National Academies of Sciences, Engineering and Medicine have launched a year-long public Consensus Study on Human Gene Editing to examine the clinical, ethical, legal, and social implications of the use of human genome editing technologies in biomedical research and medicine. Findings should be available by late 2016 and are expected to provide a worldwide framework for human gene-editing research.